Infection by many Category A, B, and C Priority Pathogens activates the innate immune system triggering an exuberant release of cytokines and other permeability factors that destabilize the endothelial barrier. The loss of vascular integrity results in non-cardiogenic edema, shock, multi-organ failure and death. We propose a new and broad therapeutic platform for reducing morbidity and mortality based on our identification of an endothelial receptor, Robo4. Activation of the Robo4 receptor by its ligand , Slit proteins, preserves the integrity of the endothelial barrier and interferes with the downstream signaling cascades from multiple permeability factors including TNF-alpha, interleukins, and thrombin in the endothelium. In animal models, Robo4 stabilizes the endothelium following diverse insults including mechanical injury, ischemia, and cytokine storm. We have recently demonstrated that in two rodent models of pathogen induced acute lung injury, this approach reduced edema, shock and death. Ultimately, our goal is to create a single platform for treating infections from a variety of Category A, B and C priority pathogens. AIM 1: Determine efficacy of Robo4 peptide agonist in rodent models of viral hemorrhagic fevers. We will use viral hemorrhagic fever models to examine whether activating Robo4 in these non-influenza viral pathogens (arenavirus hemorrhagic fever virus) reduces mortality. This Aim is facilitated by an active collaborative effort that has already established efficacy in bacterial endotoxin and H5N1 animal models. AIM 2: Optimize a Robo4 peptide agonist. We will optimize recombinant Slit-like peptide capable of activating the Robo4 signaling pathway by serial mutagenesis. Studies will focus on delivery, stability, and production requirements needed to prepare for future GMP/GLP studies. AIM 3: Robo signaling in endothelial and epithelial integrity. We will determine whether Slit-Robo4 enhances endothelial cell-cell interactions, and seek to understand the molecular mechanism. We will use this as a model to investigate whether Slit-Robo1 signaling plays a similar role in epithelial-cell-cell contacts. This project fits within the RMRCE Integrated Research Focus on Viral Therapeutics and over the course of the five year period will interact directly with RP3.1, 3.2, 2.3 and cores C, D, and F.